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Abstract

Photosynthesis is indispensable in carbon cycling and obtaining renewable carbon. Operated by cyanobacteria, algae and plants, this process provides reduced carbon and molecular oxygen, consumes atmospheric CO<sub>2</sub> and harnesses solar energy. Photosynthesis is also central to the production of biofuels. Diatoms, a class of marine algae, contribute 20% to 40% of global photosynthetic productivity despite surviving in CO<sub>2</sub>-depleted and nitrogen-limited environments. This makes diatoms ideal models to study efficient photosynthetic, specifically carbon concentrating mechanisms (CCM). It has been long debated that whether the unicellular marine diatom <italic>Phaeodactylum tricornutum</italic> operates a CCM, and whether the CCM is biophysical or biochemical (C4) in nature, with existing (circumstantial) experimental evidence divided amongst the two possibilities. Through isotope labeling experiments (ILE) and metabolic flux analysis (MFA), we provide for the first time significant, direct evidence for a biochemical CCM and the potential combined operation of a biochemical and a biophysical CCM. Additionally, we shed light on how genes regulating this complex process respond to critical environmental variables. Furthermore, we report the use of isotope-assisted metabolic flux analysis to study organic carbon (especially glucose) assimilation in <italic>P. tricornutum</italic>. Our steady state ILEs reveal glucose assimilation under light and potentially which genes may be responsible for glucose metabolism. We then studied nitrogen (mainly urea) assimilation through instationary <super>15</super>N and <super>13</super>C labeling experiments, to find indications of an unusual pathway of urea assimilation. Gene expression trends under various environmental conditions suggest the possible participation of the urea cycle in assimilating nitrogen in <italic>P. tricornutum</italic>, and how this metabolically differs from nitrate and ammonium assimilation. We anticipate that this work will not only improve understanding of unicellular C4 CCMs, but provide insights to explain the ecological success of diatoms in adapting to challenging environments.